1. Field of Invention
The present invention relates to a method for preparing a transformant lacking the selective marker gene, by site-specific recombination in yeast. The method of the invention may be used to obtain yeast transformants having no selective marker genes after introduction of a target gene into yeast.
2. Related Art
A number of gene introduction methods have been reported to date, all of which require markers for selection of recombinants because of low efficiency of gene introduction. Selective markers include those which revive prototrophy from auxotrophy when used in yeast, but usually resistance genes against drug agents such as antibiotics are used. However, the selective marker genes are preferably removed after selection of the transformants, for reasons of safety of the recombinants in practical use. Furthermore, due to the scarcity of selective markers which can be efficiently used, the marker genes are preferably reused for repeated transformation of the same individuals.
In order to overcome these problems there have been developed a few methods for removing selective marker genes from transformants. For example, in methods such as the co-transformation method, a gene to be introduced and a selective marker gene are placed on separate plasmids or DNA fragments and introduced simultaneously into a cell as separate constructs. According to this method, each of the genes exists independently and therefore it is possible to obtain individuals among the progeny which have the introduced target gene but lack the selective marker gene.
Methods utilizing transposons have also been developed. These methods rely on the action of transposons after gene introduction, to eliminate the link between the introduced target gene and the selective marker gene to allow obtainment of individuals among the progeny which have the introduced target gene but lack the selective marker gene, similar to the preceding method. However, these methods require generation of progeny, which causes the procedures to be complicated and time-consuming. In addition, variability is also produced among the progeny, thus lowering the practical usefulness.
On the other hand, methods utilizing site-specific recombination have also been developed. Site-specific recombination involves 2 elements, namely the enzyme which carries out recombination and a specific nucleotide sequence recognized by the enzyme, and recombination enzymes are known to induce recombination between 2 recognized sequences. Such recombination induces such phenomena as deletions, insertions and inversions, depending on the arrangement of recognized sequence. The four site-specific recombinants known are bacteriophage P1-derived Cre/lox, Saccharomyces cerevisiae-derived FLP/FRT, Zygosaccharomyces rouxii-derived R/RS and bacteriophage Mu-derived Gin/gix.
A great number of site-specific recombinations have been reported using these systems (Odell, J. T. and Russell, S. H., In: Paszkowski (ed.) Homologous Recombination and Gene Silencing in Plants, pp.219-270, 1994, Kluwer Academic Publishers, Netherlands; Yoder, J. I. and Goldsbrough A. P., Bio/Technology, 12, 263-267, 1994). For example, a Saccharomyces cerevisiae FLP/FRT system has been used, wherein the marker gene is removed with methylotrophic yeast (Pichia pastoris) (Cregg, J. M. and Madden, K. R., Mol. Gen. Genet. 219, 320-323, 1989).
The above authors used the ARG4 gene as a selective marker, and incorporated the ARG4 gene into a repeating FRT sequence in the same direction, to transform an arg4 mutant of methylotrophic yeast. A plasmid containing the recombinant enzyme gene FLP was then introduced into the same methylotrophic yeast to induce a site-specific recombination, by which the ARG4 selective marker gene was successfully removed. Although the ability to utilize site-specific recombination in this manner for removing markers had already been reported, as is clear from the example of Cregg et al., the method adopted for inducing the site-specific recombination is to introduce the recombination enzyme gene after the first transformants to induce the site-specific recombination.
In other words, two transformations are necessary for the induction, and therefore two separate selective markers are also necessary. Other reported site-specific recombinations also involve introduction of the recombination enzyme gene after obtaining the first transformants, and thus it is essential to introduce the recombination enzyme gene by a second transformation or by hybridization.
Araki et al. have demonstrated that a mechanism for site-specific recombination exists on the Zygosaccharomyces rouxii plasmid pSR1 (Araki, H. et al., J. Mol. Biol., 182, 191-203, 1985).
Plasmid pSR1 is a circular plasmid of 6251 bp, which is known to have a pair of inverted repeats with 959 bp in the molecule, with the site-specific recombination occurring between the inverted repeats. The recombination site in the inverted repeat consists of a 7 bp spacer sequence between short inverted repeats of 12 bp and 4 identical 12 bp sequences continue to repeat on one side. The site-specific recombination occurs when the recombination-performing enzyme (R protein) encoded in the plasmid itself binds to the R sensitive sequence, which is a specific nucleotide sequence on the recombination site in the inverted repeat.
A 31 bp sequence comprising a 7 bp-spacer portion and two 12 bp inverted repeats is known as an R sensitive sequence (RS sequence) (Matsuzaki, H. et al., Biosci. Biotech. Biochem., 58, 1632-1637, 1994). This sequence is listed as SEQ ID No.1. However, using the 31 bp R sensitive sequence for site-specific recombination is impractical since the structure after recombination includes the recognition site of the site-specific enzyme remaining in the chromosomal or plasmid DNA, which may induce unwanted recombination.